JP2007238474A - Method for eluting organic material adsorbed to sparingly water-soluble metal compound and method for purification of organic material by using said elution method - Google Patents
Method for eluting organic material adsorbed to sparingly water-soluble metal compound and method for purification of organic material by using said elution method Download PDFInfo
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本発明は、難水溶性金属化合物に吸着したポリペプチド若しくはその複合体又は核酸若しくはその複合体である有機物を効果的に溶出させる方法に関するものである。 The present invention relates to a method for effectively eluting a polypeptide adsorbed on a poorly water-soluble metal compound or a complex thereof, or a nucleic acid or an organic substance that is a complex thereof.
難水溶性金属化合物、特に元素周期表2族に属するバリウム、カルシウム及びマグネシウムの硫酸塩、リン酸塩、水酸化物及び炭酸塩、並びに典型金属元素であるアルミニウムのリン酸塩及び水酸化物は、生物学的には不活性で生理活性物質の不活化が起きにくい事が知られている。また、生物活性タンパク質や核酸との親和性が高いため、ワクチン、血液タンパク、成長ホルモン、食品添加物、インターフェロン等の精製に応用されている（特許文献１、非特許文献１〜６）。 Poorly water-soluble metal compounds, especially barium, calcium and magnesium sulfates, phosphates, hydroxides and carbonates belonging to Group 2 of the Periodic Table of Elements, and the typical metal elements aluminum phosphates and hydroxides are It is known that biologically inactive and physiologically inactive substances are hardly inactivated. Moreover, since it has high affinity with biologically active proteins and nucleic acids, it is applied to purification of vaccines, blood proteins, growth hormones, food additives, interferons, etc. (Patent Document 1, Non-Patent Documents 1 to 6).
難水溶性金属化合物に吸着するタンパク質や核酸等のような有機物の中には、該化合物に強固に吸着するものが存在する。そのような有機物を溶出させる場合には、従来、溶出液中のアルカリ金属イオン、リン酸イオンもしくはキレート剤濃度を増加させるという方法が採られているが、溶出量は十分ではなく、回収率が低くなるという問題があった。 Some organic substances such as proteins and nucleic acids adsorbed to poorly water-soluble metal compounds are strongly adsorbed to the compounds. When eluting such organic substances, a method of increasing the concentration of alkali metal ions, phosphate ions or chelating agents in the eluate has been conventionally employed, but the elution amount is not sufficient and the recovery rate is high. There was a problem of being lowered.
従って、本発明の目的は、難水溶性金属化合物を用いてタンパク質や核酸等のような有機物を精製する方法において、該有機物が吸着された難水溶性金属化合物からの該有機物の溶出量を増大させ、回収率を高めることができる手段を提供することである。 Therefore, an object of the present invention is to increase the elution amount of the organic substance from the poorly water-soluble metal compound to which the organic substance is adsorbed in a method of purifying an organic substance such as protein or nucleic acid using the hardly water-soluble metal compound. And providing a means capable of increasing the recovery rate.
本願発明者らは、鋭意研究の結果、タンパク質や核酸等のような有機物が吸着された難水溶性金属化合物から該有機物を溶出させる際に、糖類および／またはアミノ酸若しくはその塩を添加した溶出液を用いることで高い回収率が得られることを見出し、本願発明を完成した。 As a result of intensive research, the inventors of the present application have added an eluate to which a saccharide and / or an amino acid or a salt thereof is added when eluting the organic substance from a poorly water-soluble metal compound adsorbed with an organic substance such as protein or nucleic acid. The present inventors have found that a high recovery rate can be obtained by using the present invention and completed the present invention.
すなわち、本願発明は、ポリペプチド及びその複合体並びに核酸及びその複合体から成る群から選ばれる少なくとも１種の有機物が吸着された難水溶性金属化合物を溶出液と接触させて前記有機物を前記難水溶性金属化合物から溶出する方法において、該溶出液が、糖類及び／又はアミノ酸若しくはその塩を含むことを特徴とする溶出方法を提供する。また、本願発明は、そのような溶出方法により溶出工程が行なわれることを特徴とする有機物の精製方法を提供する。 That is, the present invention is directed to bringing a hardly water-soluble metal compound adsorbed with at least one organic substance selected from the group consisting of a polypeptide, a complex thereof, and a nucleic acid and a complex thereof, into contact with an eluate to make the organic substance the above difficult substance. In a method for eluting from a water-soluble metal compound, an elution method is provided in which the eluate contains a saccharide and / or an amino acid or a salt thereof. In addition, the present invention provides a method for purifying an organic substance, wherein the elution step is performed by such an elution method.
本発明の方法によれば、従来法では難水溶性金属化合物からの溶出が困難であったタンパク質又は核酸等の有機物を高い回収率で得る事が可能である。 According to the method of the present invention, it is possible to obtain an organic substance such as protein or nucleic acid, which has been difficult to elute from a hardly water-soluble metal compound by a conventional method, at a high recovery rate.
本発明の溶出方法は、タンパク質や核酸等のような有機物が難水溶性金属化合物に吸着する性質を利用して、難水溶性金属化合物を用いて前記有機物を精製する方法において、該難水溶性金属化合物に吸着した前記有機物を該難水溶性金属化合物から溶出する際の溶出方法として、特に好適に用いられる。従って、本発明は、本発明の溶出方法と共に、該溶出方法を用いた精製方法をも提供するものである。 The elution method of the present invention utilizes the property of adsorbing organic substances such as proteins and nucleic acids to the poorly water-soluble metal compound, and purifies the organic substance using the poorly water-soluble metal compound. It is particularly preferably used as an elution method when the organic substance adsorbed on the metal compound is eluted from the poorly water-soluble metal compound. Therefore, the present invention also provides a purification method using the elution method together with the elution method of the present invention.
本発明の溶出方法により溶出される有機物は、ポリペプチド及びその複合体並びに核酸及びその複合体から成る群から選ばれる少なくとも１種の有機物である。ここで、「ポリペプチド」は、２個以上のアミノ酸がペプチド結合により結合したものを意味し、タンパク質も包含される。「複合体」という語は、ポリペプチド又は核酸と、他の物質とが結合した物質を意味し、その例としてはポリペプチドの場合には糖タンパク質やリポタンパク質等、核酸の場合には核酸とタンパク質の複合体等が挙げられる。該有機物は、難水溶性金属化合物に吸着するものであれば特に限定されないが、具体例としては、酵素、抗体、膜タンパク質、ウイルス、ワクチン用抗原、ssDNA、dsDNA、RNA、プラスミドDNA等の生化学分野及び医療学分野でタンパク質又は核酸と分類されるものが挙げられる。 The organic substance eluted by the elution method of the present invention is at least one organic substance selected from the group consisting of polypeptides and complexes thereof and nucleic acids and complexes thereof. Here, the “polypeptide” means a peptide in which two or more amino acids are bound by peptide bonds, and includes proteins. The term “complex” means a substance in which a polypeptide or nucleic acid is bound to another substance. Examples thereof include glycoproteins and lipoproteins in the case of polypeptides, and nucleic acids in the case of nucleic acids. Examples include protein complexes. The organic substance is not particularly limited as long as it adsorbs to a poorly water-soluble metal compound. Specific examples thereof include enzymes, antibodies, membrane proteins, viruses, vaccine antigens, ssDNA, dsDNA, RNA, plasmid DNA, and the like. Examples include those classified as proteins or nucleic acids in the chemical and medical fields.
本発明で用いられる難水溶性金属化合物としては、元素周期表２族に属するバリウム、カルシウム及びマグネシウムの硫酸塩、リン酸塩、水酸化物及び炭酸塩、並びに典型金属元素であるアルミニウムのリン酸塩及び水酸化物が挙げられる。これらの物質の形状等は特に限定されないが、平均粒子径0.1〜300μmの粒子状とすることもできる。難水溶性金属化合物が充填されたカラム又は流動床筒を用いるカラムクロマトグラフィー法又は吸着流動床法によって目的の有機物を難水溶性化合物に吸着させる場合には、このような粒子状の難水溶性金属化合物を有利に用いることができる。 Examples of the poorly water-soluble metal compounds used in the present invention include barium, calcium and magnesium sulfates, phosphates, hydroxides and carbonates belonging to Group 2 of the Periodic Table of Elements, and aluminum phosphate which is a typical metal element Examples include salts and hydroxides. The shape and the like of these substances are not particularly limited, but may be in the form of particles having an average particle size of 0.1 to 300 μm. When adsorbing a target organic substance to a poorly water-soluble compound by a column chromatography method using a column or a fluidized bed cylinder packed with a poorly water-soluble metal compound or an adsorption fluidized bed method, such a particulate poorly water-soluble compound is used. Metal compounds can be used advantageously.
目的の有機物を難水溶性金属化合物に吸着させる方法については特に限定されないが、試料原液中に難水溶性金属化合物を投入して目的の有機物を吸着させるバッチ法、試料原液を難水溶性金属化合物が充填されたカラム又は流動床筒内に流入することにより目的の有機物を吸着させるカラムクロマトグラフィー法又は吸着流動床法等が挙げられる。 The method of adsorbing the target organic substance to the poorly water-soluble metal compound is not particularly limited, but the batch method in which the target organic substance is adsorbed by introducing the poorly water-soluble metal compound into the sample stock solution, and the sample stock solution to the poorly water-soluble metal compound Column chromatography method or adsorption fluidized bed method in which a target organic substance is adsorbed by flowing into a column or fluidized bed cylinder packed with.
溶出は、目的の有機物が吸着された難水溶性金属化合物を溶出液と接触させることにより行なわれ、例えば有機物が吸着された難水溶性金属化合物を溶出液中に浸漬すること等により行なうことができる。あるいは、カラムクロマトグラフィー法が適用される場合であれば、有機物が吸着されたカラムに溶出液を添加することにより、溶出を行なうことができる。 The elution is performed by bringing the slightly water-soluble metal compound on which the target organic substance is adsorbed into contact with the eluate, for example, by immersing the slightly water-soluble metal compound on which the organic substance is adsorbed in the eluate. it can. Alternatively, if a column chromatography method is applied, elution can be performed by adding an eluent to the column on which the organic matter is adsorbed.
本発明で用いられる溶出液は、糖類及び／又はアミノ酸若しくはその塩を含む。アミノ酸の塩としては、ナトリウム塩やカリウム塩のようなアルカリ金属塩が好ましい。該溶出液は好ましくは水溶液である。溶出液に含まれる糖類又はアミノ酸若しくはその塩は１種類でもよいし、複数でも良い。また、糖類並びにアミノ酸及びその塩を全て含んでいても良い。溶出液中の濃度は、特に限定されないが、糖類又はアミノ酸若しくはその塩のいずれか１種類を用いる場合でも複数を用いる場合でも、その合計濃度が0.1M以上であれば高い溶出効果を得ることができる。なお、特に限定されないが、合計濃度は通常3
M以下である。なお、許容される範囲内で糖類及び／又はアミノ酸若しくはその塩の濃度を高めることにより、あるいはまた後述の溶出液中に含まれ得る塩類濃度を高めることにより、目的とする有機物の溶出効率を高めることができる。本発明で用いられる溶出液のpHは、特に限定されないが、溶出対象の有機物の生化学活性を保持したい場合には、好ましくはpH５〜９、さらに好ましくはpH６〜８の中性付近とすることが望ましい。中性付近の溶出液を用いることにより、該有機物の活性を損なわずに溶出することができる。
The eluate used in the present invention contains sugars and / or amino acids or salts thereof. The amino acid salt is preferably an alkali metal salt such as sodium salt or potassium salt. The eluate is preferably an aqueous solution. The saccharide or amino acid or salt thereof contained in the eluate may be one kind or plural. Further, it may contain all of saccharides, amino acids and salts thereof. Although the concentration in the eluate is not particularly limited, a high elution effect can be obtained if the total concentration is 0.1 M or higher, regardless of whether one or more of saccharides, amino acids or salts thereof are used. it can. Although not particularly limited, the total concentration is usually 3
M or less. Increasing the elution efficiency of the target organic substance by increasing the concentration of sugars and / or amino acids or salts thereof within an allowable range, or by increasing the concentration of salts that can be contained in the eluate described later. be able to. The pH of the eluate used in the present invention is not particularly limited. However, when it is desired to maintain the biochemical activity of the organic substance to be eluted, the pH is preferably 5 to 9, more preferably about 6 to 8. Is desirable. By using an eluate near neutrality, elution can be performed without impairing the activity of the organic matter.
本発明で用いられる溶出液には、塩類をさらに含ませてもよい。ここで、塩類とは、酸と塩基の反応によって生じる塩のことを言い、塩化ナトリウム等のほか、リン酸塩、クエン酸塩などのようなpH緩衝能を有する弱酸とその共役塩基の塩又は弱塩基とその共役酸の塩も包含される。ここに例示したものに限定されず、目的や用途に合わせて適宜好ましいものを選択できる。また、塩類としては、ナトリウム塩やカリウム塩のようなアルカリ金属塩が好ましい。また、溶出液に含まれる塩類は１種類でもよいし、複数でも良い。例えば、前記のとおり溶出対象の有機物の生化学活性を保持したい場合には、中性付近でpH緩衝能を有するリン酸塩及び／又はクエン酸塩を含ませることが好ましい。該塩類の溶出液中の合計濃度は、特に限定されないが、通常、0.005Mから3M、好ましくは0.1Mから2Mの範囲である。 The eluate used in the present invention may further contain salts. Here, the salt refers to a salt produced by the reaction between an acid and a base, and in addition to sodium chloride and the like, a salt of a weak acid having a pH buffering ability such as phosphate and citrate and its conjugate base or Also included are salts of weak bases and their conjugate acids. It is not limited to what was illustrated here, A preferable thing can be selected suitably according to the objective and a use. As the salts, alkali metal salts such as sodium salts and potassium salts are preferable. Moreover, the salt contained in the eluate may be one kind or plural. For example, when it is desired to maintain the biochemical activity of the organic substance to be eluted as described above, it is preferable to include a phosphate and / or citrate having a pH buffering ability near neutrality. The total concentration of the salts in the eluate is not particularly limited, but is usually in the range of 0.005 M to 3 M , preferably 0.1 M to 2 M.
本発明で用いられる溶出液に含まれる糖類としては、単糖類、少糖類及び多糖類のほか、糖分子の官能基を置換、還元等して得られる誘導体(アミノ糖、糖アルコール、糖リン酸、ウロン酸など)も包含される。これらのうち、単糖類、少糖類及びそれらの誘導体が好ましい。溶出液に添加する糖類は、固液分離を容易にするために、好ましくは溶出液に可溶化した状態で用いる。すなわち、溶出液に可溶の糖類が好ましく、例えば溶出液が水溶液であれば水溶性の糖類を用いることが好ましい。好ましい糖類の具体例としては、七炭糖のセドヘプツロース、六炭糖のグルコース、フルクトース、リブロース、ガラクトース、タロース、アロース、マンノース、五炭糖のアピオース、リボース、アラビノース、キシロース、二糖のマルトース、ラクトース、スクロース、可溶性の多糖類、少糖(オリゴ糖)のフルクトオリゴ糖、ガラクトオリゴ糖、乳果オリゴ糖、デオキシ糖のデオキシリボース、フコース、ラムノース、ウロン酸のアスコルビン酸(ビタミンC)、グルクロン酸、ガラクツロン酸、アミノ糖のグルコサミン、N-アセチルガラクトサミン、N-アセチルグルコサミン、糖アルコールのグリセリン、キシリトール、ソルビトール、糖リン酸のグルコース1-リン酸、グルコース6-リン酸、フルクトース1,6-ビスリン酸および5-ホスホリボシル1-ピロリン酸などが挙げられるが、これらに限定されない。 The saccharides contained in the eluate used in the present invention include monosaccharides, oligosaccharides and polysaccharides, as well as derivatives obtained by substituting or reducing functional groups of sugar molecules (amino sugars, sugar alcohols, sugar phosphates). , Uronic acid, and the like). Of these, monosaccharides, oligosaccharides and derivatives thereof are preferred. The saccharide added to the eluate is preferably used in a state solubilized in the eluate in order to facilitate solid-liquid separation. That is, saccharides soluble in the eluate are preferable. For example, if the eluate is an aqueous solution, water-soluble saccharides are preferably used. Specific examples of preferred saccharides include heptose sugar sedoheptulose, hexose sugar glucose, fructose, ribulose, galactose, talose, allose, mannose, pentose apiose, ribose, arabinose, xylose, disaccharide maltose, lactose , Sucrose, soluble polysaccharide, oligosaccharide fructooligosaccharide, galactooligosaccharide, dairy oligosaccharide, deoxysugar deoxyribose, fucose, rhamnose, uronic acid ascorbic acid (vitamin C), glucuronic acid, galacturon Acid, amino sugar glucosamine, N-acetylgalactosamine, N-acetylglucosamine, sugar alcohol glycerin, xylitol, sorbitol, sugar phosphate glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-bisphosphate and 5-phosphoribosyl Examples thereof include, but are not limited to, 1-pyrophosphate.
本発明で用いられる溶出液に含まれるアミノ酸又はその塩は、固液分離を容易にするために、溶出液に可溶化した状態で用いることが望ましい。すなわち、溶出液に可溶のアミノ酸又はその塩が好ましく、例えば溶出液が水溶液であれば親水性アミノ酸又はその塩を用いることが好ましい。具体例としては、塩基性アミノ酸のアルギニン、リジン、ヒスチジン、酸性アミノ酸のグルタミン酸、アスパラギン酸およびそれらのアミドであるグルタミン、アスパラギン、中性アミノ酸のグリシン、トレオニン、セリンなど、並びにこれらの塩が挙げられるが、これらに限定されない。なお、前記のとおり、溶出液のpHを中性付近として用いることが望まれる場合には、溶出液に添加するアミノ酸又はその塩としては、中性付近で陽イオン又は陰イオンの存在比率の高い塩基性アミノ酸若しくは酸性アミノ酸またはそれらの塩が好ましく用いられ得る。 The amino acid or salt thereof contained in the eluate used in the present invention is desirably used in a state solubilized in the eluate in order to facilitate solid-liquid separation. That is, an amino acid or a salt thereof that is soluble in the eluate is preferable. For example, if the eluate is an aqueous solution, it is preferable to use a hydrophilic amino acid or a salt thereof. Specific examples include basic amino acids arginine, lysine, histidine, acidic amino acids glutamic acid, aspartic acid and their amides glutamine, asparagine, neutral amino acids glycine, threonine, serine, and the like, and salts thereof. However, it is not limited to these. As mentioned above, when it is desired to use the pH of the eluate as near neutral, the amino acid or salt thereof added to the eluate has a high ratio of cations or anions near neutral. Basic amino acids or acidic amino acids or salts thereof can be preferably used.
難水溶性金属化合物に吸着させた有機物を溶出させる前に、糖類、アミノ酸及びその塩、並びに塩類から成る群から選ばれる少なくとも１種以上の物質を前記溶出液よりも低濃度で含む前処理液を用いて処理してもよい。この処理工程により、試料中に存在する目的とする有機物以外の夾雑物等を可能な限り排除することができる。塩類濃度、糖類濃度、アミノ酸若しくはその塩の濃度は直線勾配又は曲線勾配のいずれかを用いて増加させるものであってもよい。この工程により、難水溶性金属化合物に吸着した物質のうち、吸着力の弱い物質が溶出されるため、目的とする有機物と夾雑物とを選択的に分離することが可能である。 A pretreatment liquid containing at least one substance selected from the group consisting of saccharides, amino acids and salts thereof, and salts at a lower concentration than the eluate before eluting the organic substance adsorbed on the poorly water-soluble metal compound. You may process using. By this treatment step, impurities other than the target organic substance present in the sample can be eliminated as much as possible. The salt concentration, saccharide concentration, amino acid or salt concentration thereof may be increased using either a linear gradient or a curve gradient. By this step, among the substances adsorbed on the poorly water-soluble metal compound, the substance having a weak adsorbing power is eluted, so that it is possible to selectively separate the target organic substance and impurities.
さらに、本発明の溶出方法は、ポリペプチド及びその複合体並びに核酸及びその複合体から成る群から選ばれる少なくとも１種の精製すべき有機物を含む原液を難水溶性金属化合物と接触させる第１工程と、該難水溶性金属化合物を前記原液から分離する第２工程と、分離した前記難水溶性金属化合物を溶出液と接触させて前記有機物を前記難水溶性金属化合物から溶出する第３工程とを含む有機物の精製方法において、第３工程を行なう方法として用いることができる。すなわち、本発明は、前記の精製方法において、前記第３工程が本発明の溶出方法により行なわれることを特徴とする有機物の精製方法をも提供する。 Furthermore, the elution method of the present invention is a first step of bringing a stock solution containing at least one organic substance to be purified selected from the group consisting of a polypeptide and a complex thereof and a nucleic acid and a complex thereof into contact with a poorly water-soluble metal compound. A second step of separating the hardly water-soluble metal compound from the stock solution, and a third step of bringing the separated hardly water-soluble metal compound into contact with an eluent to elute the organic matter from the hardly water-soluble metal compound. Can be used as a method of performing the third step. That is, the present invention also provides a method for purifying an organic substance, characterized in that, in the purification method, the third step is performed by the elution method of the present invention.
前記第１工程では、精製すべき有機物を難水溶性金属化合物に吸着させる。吸着させる方法については特に限定されないが、試料原液中に難水溶性金属化合物を投入して精製すべき有機物を吸着させるバッチ法、試料原液を難水溶性金属化合物が充填されたカラム又は流動床筒内に流入することにより精製すべき有機物を吸着させるカラムクロマトグラフィー法又は吸着流動床法等が挙げられる。カラムクロマトグラフィー法及び吸着流動床法は多量処理、再現性に優れており、本発明の精製方法の一応用例として好ましい。なお、前述したとおり、難水溶性金属化合物を平均粒子径0.1〜300μｍの粒子状とした場合には、カラムクロマトグラフィー法及び吸着流動床法を適用する際に有利である。 In the first step, the organic matter to be purified is adsorbed on the poorly water-soluble metal compound. The adsorption method is not particularly limited, but a batch method in which a slightly water-soluble metal compound is introduced into a sample stock solution to adsorb organic matter to be purified, a column or fluidized bed cylinder in which the sample stock solution is packed with a hardly water-soluble metal compound. Examples thereof include a column chromatography method or an adsorption fluidized bed method in which an organic substance to be purified is adsorbed by flowing into the inside. The column chromatography method and the adsorption fluidized bed method are excellent in mass processing and reproducibility, and are preferable as an application example of the purification method of the present invention. As described above, when the poorly water-soluble metal compound is in the form of particles having an average particle size of 0.1 to 300 μm, it is advantageous when applying the column chromatography method and the adsorption fluidized bed method.
続く第２工程では、第１工程で用いた難水溶性金属化合物を前記原液から分離する。バッチ法が適用される場合、一般的に濾過法、遠心分離法、傾斜法等が用いられるが、難水溶性金属化合物として磁性吸着剤を用いた場合は、磁気分離法を用いることができる。カラムクロマトグラフィー法が適用される場合、カラムを洗浄し、カラムに吸着した精製すべき有機物以外の物質を除去する工程が該第２工程に相当する。 In the subsequent second step, the poorly water-soluble metal compound used in the first step is separated from the stock solution. When the batch method is applied, a filtration method, a centrifugal separation method, a gradient method, or the like is generally used. However, when a magnetic adsorbent is used as the poorly water-soluble metal compound, a magnetic separation method can be used. When the column chromatography method is applied, the step of washing the column and removing substances other than the organic matter to be purified adsorbed on the column corresponds to the second step.
続く第３工程では、本発明の溶出方法により、難水溶性金属化合物に吸着した有機物の溶出が行なわれる。例えば、バッチ法が適用される場合、有機物が吸着された難水溶性金属化合物を本発明で用いられる溶出液中に浸漬することにより有機物を溶出することができる。カラムクロマトグラフィー法が適用される場合、有機物が吸着されたカラムに本発明で用いられる溶出液を添加することにより、有機物を溶出することができる。 In the subsequent third step, the organic substance adsorbed on the poorly water-soluble metal compound is eluted by the elution method of the present invention. For example, when the batch method is applied, the organic matter can be eluted by immersing the poorly water-soluble metal compound adsorbed with the organic matter in the eluent used in the present invention. When the column chromatography method is applied, the organic substance can be eluted by adding the eluent used in the present invention to the column on which the organic substance is adsorbed.
また、前記第２工程と第３工程の間に、上記した通り、糖類、アミノ酸及びその塩、並びに塩類から成る群から選ばれる少なくとも１種以上の物質を前記溶出液よりも低濃度で含む前処理液で処理する工程をさらに含ませてもよい。この工程により、前記原液中に存在する精製すべき有機物以外の夾雑物等を可能な限り排除することができ、有機物の純度を高めることができるため好ましい。 In addition, before the second step and the third step, as described above, before containing at least one substance selected from the group consisting of sugars, amino acids and salts thereof, and salts at a lower concentration than the eluate. You may further include the process processed with a process liquid. This step is preferable because impurities other than the organic matter to be purified existing in the stock solution can be eliminated as much as possible, and the purity of the organic matter can be increased.
以下、本発明の実施例に基づき具体的に説明する。ただし、本発明は下記実施例に限定されるものではない。 The present invention will be specifically described below based on examples of the present invention. However, the present invention is not limited to the following examples.
下記に示す手順にてインフルエンザウイルスの回収を行った。硫酸バリウムからなる難水溶性金属化合物を吸着担体として用いたバッチ法にて行った。 Influenza virus was collected according to the procedure shown below. This was carried out by a batch method using a poorly water-soluble metal compound composed of barium sulfate as an adsorption carrier.
[溶出液]
溶出液は以下9種類を準備した。
従来の方法の溶出液１：
12%クエン酸ナトリウム-6%塩化ナトリウム溶出液(pH 7.5)
本発明の方法の溶出液２：
溶出液１に、単糖のグルコースを1M濃度になるように可溶化した溶液(pH7.3)。
本発明の方法の溶出液３：
溶出液１に、二糖のトレハロースを1M濃度になるように可溶化した溶液(pH7.3)。
本発明の方法の溶出液４：
溶出液１に、二糖のマルトースを1M濃度になるように可溶化した溶液(pH7.2)。
本発明の方法の溶出液５：
溶出液１に、二糖のスクロースを1M濃度になるように可溶化した溶液(pH7.5)。
本発明の方法の溶出液６：
溶出液１に、糖アルコールのソルビトールを1M濃度になるように可溶化した溶液(pH7.5)。
本発明の方法の溶出液７：
溶出液１に、塩基性アミノ酸のアルギニン(塩酸塩) を1M濃度になるように可溶化した溶液(pH6.9)。
本発明の方法の溶出液８：
溶出液１に、酸性アミノ酸のグルタミン酸(ナトリウム塩) を1M濃度になるように可溶化した溶液(pH7.2)。
本発明の方法の溶出液９：
溶出液１に、中性アミノ酸のグリシンを1M濃度になるように可溶化した溶液(pH7.2)。
[Eluate]
The following 9 types of eluates were prepared.
Conventional eluate 1:
12% sodium citrate-6% sodium chloride eluent (pH 7.5)
Eluent 2 of the method of the present invention:
A solution (pH 7.3) obtained by solubilizing monosaccharide glucose in eluate 1 so as to have a concentration of 1M.
Eluent 3 of the method of the present invention:
A solution (pH 7.3) in which the disaccharide trehalose was solubilized in eluate 1 to a concentration of 1M.
Eluent 4 of the method of the present invention:
In Eluate 1, a solution (pH 7.2) in which maltose of disaccharide was solubilized to a concentration of 1M.
Eluent 5 of the method of the present invention:
In eluate 1, a solution (pH 7.5) in which disaccharide sucrose was solubilized to a concentration of 1M.
Eluent 6 of the method of the present invention:
In eluate 1, a solution (pH 7.5) in which sugar alcohol sorbitol was solubilized to a concentration of 1M.
Eluent 7 of the method of the present invention:
A solution (pH 6.9) of basic amino acid arginine (hydrochloride) solubilized in eluate 1 to a concentration of 1M.
Eluent 8 of the method of the present invention:
In eluate 1, a solution (pH 7.2) in which glutamic acid (sodium salt) of an acidic amino acid was solubilized to a concentration of 1M.
Eluent 9 of the method of the present invention:
A solution (pH 7.2) in which the neutral amino acid glycine was solubilized in the eluate 1 to a concentration of 1M.
［実験方法］
従来の方法の溶出液１および本発明方法の溶出液２〜９を用いて以下の通りバッチ法によるインフルエンザウイルスの精製回収を行い、インフルエンザウイルスの回収率(% CCA)を求めた。
[experimental method]
Using the eluate 1 of the conventional method and the eluates 2 to 9 of the method of the present invention, the influenza virus was purified and collected by the batch method as follows, and the influenza virus recovery rate (% CCA) was determined.
発育鶏卵で培養されたH3N2 A/New York株インフルエンザウイルスを含有する原料40mL (尿膜腔液 : 0.45μmフィルター処理済み)に、シュウ酸カリウム40mgを添加後、室温で15分間攪拌した。次に難水溶性金属化合物である硫酸バリウム800mgを徐々に添加し、更に室温で30分間攪拌した。 40 mg of potassium oxalate was added to 40 mL of a raw material containing H3N2 A / New York strain influenza virus cultured in growing chicken eggs (allantoic fluid: 0.45 μm filtered), and then stirred at room temperature for 15 minutes. Next, 800 mg of barium sulfate, which is a poorly water-soluble metal compound, was gradually added and further stirred at room temperature for 30 minutes.
遠心処理(2000rpm,10分,4℃)後、吸着上清を傾斜法により除去し、沈渣に溶出液を20mL添加し、室温で30分間攪拌した。続く遠心処理(2000rpm,10分,4℃)後、溶出液を傾斜法により回収した。 After centrifugation (2000 rpm, 10 minutes, 4 ° C.), the adsorption supernatant was removed by a gradient method, 20 mL of the eluate was added to the sediment, and the mixture was stirred at room temperature for 30 minutes. After subsequent centrifugation (2000 rpm, 10 minutes, 4 ° C.), the eluate was collected by a gradient method.
得られた溶出液から2.5mLを採取し、セファデックス G-25（商品名）脱塩カラムで1mLずつ分画処理(移動層:10mM PBS(pH7.2))した。各分画中のインフルエンザウイルス生物活性を赤血球凝集素(HA)価より調べ、HA陽性の5分画をまとめた。このように脱塩処理した溶出液由来の検体と、同様に脱塩処理を行った原料のCCA価を比較することにより、溶出液を用いたインフルエンザウイルスの回収率(% CCA)を求めた。 2.5 mL was collected from the obtained eluate, and fractionated by 1 mL using a Sephadex G-25 (trade name) desalting column (mobile layer: 10 mM PBS (pH 7.2)). Influenza virus biological activity in each fraction was examined from the hemagglutinin (HA) titer, and 5 HA positive fractions were collected. The recovery rate (% CCA) of influenza virus using the eluate was determined by comparing the CCA titer of the desalted eluate-derived specimen with the same desalted raw material.
[結果]
従来の方法の溶出液１および本発明方法の溶出液２〜９を用いた実験結果を下表に示す。通常の溶出液に、糖類ではグルコース、トレハロース、マルトース、スクロース、ソルビトール、アミノ酸ではアルギニン(塩酸塩)、グリシン、グルタミン酸（ナトリウム塩）を各々添加することによりインフルエンザウイルス回収率が90%以上となった。従来の溶出液による回収率が50%未満であったことから、本発明法は従来法と比較して明らかに溶出効果が優れていることがわかる。
[result]
The experimental results using the eluent 1 of the conventional method and the eluates 2 to 9 of the method of the present invention are shown in the table below. By adding glucose, trehalose, maltose, sucrose, sorbitol, and arginine (hydrochloride), glycine, and glutamic acid (sodium salt) for amino acids to the normal eluate, the influenza virus recovery rate exceeded 90%. . Since the recovery rate by the conventional eluate was less than 50%, it can be seen that the method of the present invention is clearly superior to the conventional method in the elution effect.
Claims (14)
Between the second step and the third step, a step of treating with a pretreatment liquid containing at least one substance selected from the group consisting of sugars, amino acids and salts thereof, and salts at a lower concentration than the eluate. 14. The method of claim 13, further comprising:
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| CN102604640A (en) * | 2011-01-19 | 2012-07-25 | 广州合诚三先生物科技有限公司 | Method for comprehensively extracting rosmarinus officinalis and preparing fat-soluble instant antioxidant |
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| CN102604640A (en) * | 2011-01-19 | 2012-07-25 | 广州合诚三先生物科技有限公司 | Method for comprehensively extracting rosmarinus officinalis and preparing fat-soluble instant antioxidant |
| CN102604640B (en) * | 2011-01-19 | 2014-02-26 | 广州合诚三先生物科技有限公司 | Method for comprehensively extracting rosmarinus officinalis and preparing fat-soluble instant antioxidant |
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